Mutations in the gene encoding presenilin-1 (PS1) protein are causative in the majority of early-onset familial Alzheimer's disease (FAD). Thus, a central focus of Alzheimer's research is to resolve the physiological role of PS1 in normal and PS1-linked FAD brain. Recent studies show that PS1 is a transmembrane protein confined to intracellular membrane compartments, where it undergoes endoproteolytic cleavage into N- and C-terminal fragments. The principal goal of this research is to utilize the specificity of monoclonal antibodies recognizing N- and C-terminal domains of PS1 and a receptor epitope fused to serial truncations of PS1 to address two fundamental aspects of PS1 biology. First, an epitope protection assay and complementary immunogold-EM study will be adapted to determine the transmembrane topology of wild type PS1. Secondly, immunogold-EM will be employed to define the precise ultrastructural localization of processed PS1 derivatives. Taken together, these studies will be critical to understanding the subcellular interactions of PS1 with other molecules along the functional pathways of PS1 and along the structure of the protein itself. We will also examine the effects of select PS1 mutations on wild type topology and native N- and C-terminal fragment distribution to explore potential sources of mutant PS1 malfunction.